The Hypoxic Tumour Microenvironment, Patient Selection and Hypoxia-modifying Treatments

Clinical Oncology (2007) 19: 385e396 doi:10.1016/j.clon.2007.03.001

Overview

The Hypoxic Tumour Microenvironment, Patient Selection and Hypoxia-modifying Treatments I. J. Hoogsteen*, H. A. M. Marresy, A. J. van der Kogel*, J. H. A. M. Kaanders* *Department of Radiation Oncology, Radboud University, Nijmegen Medical Centre, Nijmegen, The Netherlands; yDepartment of Otorhinolaryngology/Head and Neck Surgery, Radboud University, Nijmegen Medical Centre, Nijmegen, The Netherlands

ABSTRACT: Tumour hypoxia has been found to be a characteristic feature in many solid tumours. It has been shown to decrease the therapeutic efficacy of radiation treatment, surgery and some forms of chemotherapy. Successful approaches have been developed to counteract this resistance mechanism, although usually at the cost of increased short- and long-term sideeffects. New methods for qualitative and quantitative assessment of tumour oxygenation have made it possible to establish the prognostic significance of tumour hypoxia. The ability to determine the degree and extent of hypoxia in solid tumours is not only important prognostically, but also in the selection of patients for hypoxia-modifying treatments. To provide the best attainable quality of life for individual patients it is of increasing importance that tools be developed that allow a better selection of patients for these intensified treatment strategies. Several genes and proteins involved in the response to hypoxia have been identified as potential candidates for future use in predictive assays. Although some markers and combinations have shown potential benefit and are associated with treatment outcome, their clinical usefulness needs to be validated in prospective trials. A review of published studies was carried out, focusing on the assessment of tumour hypoxia, patient selection and the possibilities to overcome hypoxia during treatment. Hoogsteen, I. J. et al. (2007). Clinical Oncology 19, 385e396 ª 2007 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. Key words: Hypoxia, modification, patient selection

Introduction The response to cancer treatment is largely influenced by the tumour microenvironment. One of the most important factors playing a crucial role in this process is the existence of hypoxia in solid tumours. It has been shown to decrease the therapeutic efficacy of radiation treatment, surgery and some forms of chemotherapy. In 1955 it was first recognised that human tumours contain regions of hypoxic cells and that the radiocurability of these tumours was limited by hypoxia [1]. With the introduction in the late 1980s of a computerised polarographic needle electrode system, it became possible to measure tumour oxygenation status [2]. It enabled the rapid identification and characterisation of tumour hypoxia and the assessment of its clinical relevance. Hypoxia was found to be a characteristic feature in about 50% of all locally advanced solid tumours, irrespective of their size and histology [3]. Furthermore, clinical studies in carcinomas of the uterine cervix [4,5] and head and neck [6,7] showed a correlation between hypoxia and a poor response to radiotherapy. Hypoxic tumours may also be less responsive to chemotherapeutic agents. This has been shown in vitro and in vivo in a variety of tumours [8e10]. Oxygen deprivation induces changes in a variety of 0936-6555/07/190385þ12 $35.00/0

cellular processes, such as apoptosis, proliferation and repair, leading to treatment resistance [11]. The causes of hypoxia are multifactorial and include abnormal and chaotic tumour vasculature, impaired blood perfusion, rate of oxygen consumption and anaemia [3]. Severe tumour hypoxia ultimately leads to tissue necrosis, but non-lethal levels of hypoxia may have a strong effect on tumour cell biology. It elicits multiple cellular response pathways that alter gene expression and lead to proteomic changes. These effects, in turn, are capable of promoting increased metastasis, angiogenesis and the selection of cells with diminished apoptotic potential, leading to a worse outcome. Hypoxia may thus provide an overall positive advantage for malignant growth [12,13]. Several therapeutic approaches have been developed to overcome hypoxia, some being successful and others not. The main focus has been on either eliminating hypoxia by breathing a high oxygen gas mixture such as carbogen [14] and the use of hyperbaric oxygen [15] or by sensitising hypoxic cells to radiation with hypoxic cell sensitisers [16]. Newer approaches, such as gene therapy or the inhibition of response pathways, are currently under investigation. For these treatments to be successful it is important to develop predictive assays reflecting the biological heterogeneity of

ª 2007 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

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individual tumours, thereby providing a tool to optimise and better select patients for those treatment strategies. This review summarises the approaches that have been undertaken to counteract tumour hypoxia and highlights the importance of understanding hypoxic response pathways.

Pathophysiology of Tumour Hypoxia The presence of hypoxic regions is a characteristic pathophysiological property of many solid tumours and has been found in a wide range of human malignancies. It arises as a result of an imbalance between the supply and consumption of oxygen [17e19]. Several major pathogenetic mechanisms are involved, such as abnormal tumour vasculature, limited tissue perfusion and tumour-associated or therapyassociated anaemia leading to a reduced oxygen transport capacity of the blood [3,17]. Tumour hypoxia can be divided into two separate categories, acute and chronic hypoxia, although the distinction is rather artificial. Not only a mixture of both types of hypoxia can be found in a tumour, but also regions of intermediate hypoxia. Perfusion-limited or acute hypoxia is often transient and may be due to severe structural and functional abnormalities of the tumour microvessels [18]. These abnormalities cause disturbances in the blood supply, leading to a temporal shut down of vessels, decreasing gradients of oxygen and nutrients and even reversion of blood flow [18,20,21]. Hypoxia can also be caused by an increase in diffusion distances, often resulting in diffusion-limited or chronic hypoxia, leaving cells deprived of oxygen and other nutrients [18]. Oxygen measurements in locally advanced primary tumours have shown the heterogeneous distribution of oxygen levels rather than two large populations of aerobic and hypoxic cells. They revealed a median partial pressure of oxygen (pO2) of 8 mmHg, with 27.5% below 2.5 mmHg and 40% below 5 mmHg [18,22]. It is believed that due to these differences in oxygenation status and the subsequent adaptation of tumour cells to hypoxia, an overall positive advantage to tumour growth and treatment resistance may result.

Tumour Hypoxia and Treatment Resistance The most extensive research on the contribution of hypoxia to treatment resistance has been carried out in radiation oncology, where adequate intratumoral oxygen levels are required to be maximally cytotoxic. The biological effect of radiation depends on the degree of tissue oxygenation, and hypoxic cells are about three-fold more resistant to radiation than well-oxygenated cells [23]. Hypoxiamediated resistance to radiation therapy is multifactorial, involving a variety of mechanisms. Under physiological conditions when radiation is absorbed in tissues, free radicals are produced. These highly reactive but shortlived free radicals produce double-strand breaks in DNA leading to cell death. The presence of oxygen can stabilise (‘fix’) the free radicals, leading to a further increase in DNA damage and reducing the ability to repair the damage. Oxygen is therefore essential for irradiation to be effective

[17,24,25]. Indirectly, hypoxia-induced genetic and proteomic changes may have a substantial effect on radiation resistance by altering proliferation kinetics, cell-cycle position, inhibiting apoptosis, regulation of angiogenesis and changing cellular metabolism by increasing anaerobic glycolysis [10,12]. Oxygen dependency has also been documented for chemotherapy and surgery, although the exact mechanisms are still unclear [8,9,26]. A poor and fluctuating blood flow, as well as increased diffusion distances, may indirectly result in diminished distribution of chemotherapeutic agents, and reduced cellular proliferation and tissue acidosis may also play a role [10,25]. Radiosensitivity is progressively reduced when the pO2 in a tumour is less than 25e30 mmHg [17]. Overall, the critical pO2 in tumours, below which cellular changes associated with hypoxia have been observed, is 8e10 mmHg [17]. Many classical radiobiological studies have shown that cells with pO2 ! 0.5 mmHg are maximally resistant to the lethal effects of irradiation. However, it was suggested by Wouters and Brown [22] that cells at intermediate oxygen levels between 0.5 and 20 mmHg could be more important in determining the response to fractionated radiotherapy. It is thought that these cells have a greater chance to retain their viability and survive irradiation. Those cells may form an important subpopulation in the tumour able to proliferate under intermediate hypoxic conditions. Lately, interest has focused on the existence of transient hypoxia. Temporal fluctuations in tumour oxygenation, due to changes in blood flow, may lead to acute hypoxia [27,28]. Importantly, as the oxygenation status of tumour cells differs over time, there are concomitant changes in their sensitivity to radiation and chemotherapy and also in their proliferative potential. These fluctuations and temporary protective effects during transient hypoxic conditions may have a great effect on treatment efficacy and this area deserves further study [27].

Tumour Hypoxia and Anaemia Anaemia is a common condition in cancer patients that may be related to treatment or the malignant disease itself. Clinical trials have shown a significant association between low haemoglobin levels and poor outcome of both radiation therapy and chemotherapy in various solid tumours [29,30]. It was suggested that a decrease in the oxygen-carrying capacity of the blood caused by anaemia might be a major causative factor for the development of hypoxia and reduced therapeutic efficacy. This could be due to the fact that normal tissues are able to compensate mild to moderate anaemia by increasing tissue perfusion, whereas these compensatory mechanisms are reduced in tumours due to abnormal vasculature [31]. The apparent relationship between anaemia, tumour hypoxia and outcome has been investigated in vitro and in vivo [31e35]. In a rat model, Kelleher et al. [33] showed that tumour-related anaemia resulted in substantial worsening of tumour oxygenation measured with polarographic needle electrodes. The correction of anaemia with either a blood transfusion or the administration of recombinant

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human erythropoietin (rhEPO) only partially led to a reduction in tumour hypoxia. In the clinical setting, however, the influence of haemoglobin level on tumour oxygenation is not clear so far, with contradictory findings from different studies. Some have shown a significant association between haemoglobin level and tumour oxygenation measured with polarographic needle electrodes [31,34,35], whereas others could not find any correlation [4,36]. Also, on the molecular level, no association between haemoglobin level and the upregulation of hypoxia-inducible proteins, such as hypoxiainducible factor 1a (HIF-1a), microvessel density, or the activation of angiogenic pathways, such as vascular endothelial growth factor, could be shown [37,38]. Although it is obvious that anaemia and hypoxia are important factors influencing treatment outcome, the relationship between both remains rather controversial. Nevertheless, there is evidence suggesting that the correction of anaemia may enhance radiosensitivity and chemosensitivity of solid tumours, supporting the proposed association [33]. Thus, the correction of anaemia with either EPO or a blood transfusion could, in principle, be a valuable strategy to improve therapeutic and patient outcomes. However, results from clinical trials have not been conclusive about the use of rhEPO or blood transfusions and more research is needed to determine their clinical usefulness.

Hypoxia Modification in Radiotherapy With the evidence that tumour hypoxia is a characteristic feature and of prognostic significance in patients with carcinoma of the head and neck and the uterine cervix, several treatment modifications have been tested in the clinic. Although a number of trials did not show any benefit, an overview analysis clearly showed that the modification of tumour hypoxia significantly improved radiotherapy outcome, especially in head and neck carcinomas [39]. However, despite these positive data, hypoxic modification is still not generally accepted in the clinic. Different mechanisms, summarised in Table 1, form the basis of counteracting tumour hypoxia. They have been investigated earlier or are currently under investigation in phase III trials.

Hyperbaric Oxygen and Accelerated Radiotherapy Combined with Carbogen and Nicotinamide A straightforward approach to reduce hypoxia is by increasing the oxygen supply to tumour cells. The use of hyperbaric oxygen was rapidly introduced into the clinic and resulted in a significant improvement in local tumour control in carcinoma of the head and neck and the uterine cervix [15]. However, the delivery of radiation therapy in hyperbaric oxygen is a demanding and complex technique, and an increased incidence of late radiation morbidity was observed [40]. For these and other reasons, hyperbaric oxygen is not generally accepted in daily practice. A more feasible alternative is the use of the hyperoxic gas carbogen (95e98% O2 þ 2e5% CO2). A new treatment approach,

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which is currently being evaluated in a phase III trial, is accelerated radiotherapy combined with carbogen and nicotinamide (ARCON). ARCON combines accelerated fractionated radiotherapy (to counteract repopulation) with the hyperoxic gas carbogen (to reduce diffusion-limited hypoxia) and nicotinamide, a vasoactive agent (for the reduction of perfusion-limited hypoxia) [41]. This approach was developed in the early 1990s. With both conventional and accelerated schedules of radiotherapy, large and significant increases in radiosensitisation were observed when using carbogen and nicotinamide [42]. Furthermore, in xenograft tumour lines, carbogen breathing was found to be very effective in reducing diffusion-limited hypoxia [43]. Phase I and II trials using ARCON have been started, with the largest ones in head and neck cancer, bladder cancer and glioblastoma [14,44e46]. In these types of cancer, the existence of hypoxia as a potential mechanism of radioresistance has been shown, with the most abundant evidence for head and neck carcinomas [6,47,48]. The largest clinical experience with ARCON thus far is from a phase II trial in 215 patients with head and neck squamous cell carcinomas. High locoregional tumour control rates were observed for tumours of the larynx (77%) and the oropharynx (72%) (Fig. 1) [49]. The results from this study support the concept of increased susceptibility of tumours to the biologically based approach of ARCON, which offers excellent opportunities for organ preservation. Recently, however, a phase III trial in head and neck squamous cell carcinomas could not show a significant improvement in local tumour control with the addition of carbogen breathing to radiotherapy [50]. The size of this study was limited, however, and more and larger randomised trials are needed to reach definite conclusions. Bladder cancer is another tumour type where organ preservation treatment offers an important advantage over surgery. A phase II trial in bladder cancer indicated improvements in local control and survival with ARCON [45]. In the head and neck carcinomas there was an increase in acute radiotherapy-related toxicity with ARCON, but severe late morbidity was not significantly elevated for both types of cancer [14,45,51]. These studies formed the basis for further study of the potential of ARCON and, currently, phase III trials with ARCON for laryngeal and bladder cancer are ongoing in the Netherlands and the UK.

Hypoxic Cell Radiosensitisers A great deal of research has been dedicated to the development of compounds that could mimic oxygen and preferentially sensitise hypoxic cells to radiation. These are nitroimidazole compounds, and one of the first and most widely tested agents was misonidazole. Many trials have been conducted, with some showing good effectiveness, whereas others could not find any benefit. The major limitation of misonidazole proved to be severe and doselimiting neuropathy [40,52]. This stimulated the development of newer generation drugs with less toxicity, the most successful one being nimorazole. The Danish Head and Neck Cancer Study group (DAHANCA) showed a highly significant

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Table 1 e Therapeutic mechanisms to overcome hypoxia Aim Increasing oxygen supply

Treatment strategy

Currently

ARCON (carbogen and nicotinamide)

In a phase III trial for carcinomas of the larynx and bladder Has been investigated in a phase III trial, but is currently not in use for this purpose Has been tested in phase III trials. Results have been contradictory and inconclusive. Needs to be investigated Has been tested in a phase III trial, but is currently only in practice in Denmark In a phase III trial, no results have been published Has only been investigated in laboratory settings, no results from clinical trials yet Under investigation in a phase III trial Under investigation in phase I and II trials

Hyperbaric oxygen

Blood transfusion/erythropoietin

Mimic oxygen

Hypoxic cell sensitisers (nimorazole)

Targeting hypoxic cells

Hypoxic cytotoxins (tirapazamine) Gene therapy

Targeting tumour microenvironment

Anti-angiogenesis (VEGF) Inhibiting biological response pathways (HIF-1, MAPK, PI3K)

References [14,45] [15]

[33e35,123,124]

[16] [55,56,58] [53]

[53] [59e61]

ARCON, accelerated radiotherapy combined with carbogen and nicotinamide; VEGF, vascular endothelial growth factor; HIF-1, hypoxiainducible factor-1; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase.

beneficial tumour response with this drug in supraglottic laryngeal and pharyngeal tumours when combined with a conventional radiotherapy schedule [16]. Furthermore, the drug-related toxicity was limited. However, this study did not lead to the general use of nimorazole in the clinic, as it was overshadowed by negative studies with older generations of sensitisers, which were more toxic. Currently, nimorazole is only incorporated in the standard treatment of patients with head and neck cancer in Denmark.

Targeting Hypoxic Cells Another approach to overcome hypoxia is by specifically targeting hypoxic cells with bioreductive drugs or by using gene therapy [53,54]. Bioreductive drugs are compounds 100

Loco-regional control

90 80 70 60

that are reduced by biological enzymes to their toxic and active metabolites. They are designed in such a way that this metabolism occurs preferentially in the absence of oxygen. The first hypoxic cytotoxin introduced in the clinic was tirapazamine. In experimental tumours it has been shown to potentiate cell killing by ionising radiation and chemotherapeutic agents such as cisplatin [55,56]. The results from randomised phase II and III trials indicated that tirapazamine, when used in combination with radiotherapy and/or cisplatin, could improve the outcome in patients with head and neck cancer and non-small cell lung cancer, respectively [57,58]. Further clinical investigations are required to assess the potential of tirapazamine and to estimate the magnitude of gain when this drug is given in combination with radiotherapy and chemotherapy. The development of clinical protocols to overcome hypoxia based on gene therapy has gained increased interest. However, approaches that have been developed to date have not been successful, due to several deficiencies. Whether the transfer of genetic material specifically to the tumour site will have sufficient therapeutic efficacy is still under investigation.

50 40

Targeting Hypoxia Response Pathways

30 Larynx Hypopharynx Oral cavity Oropharynx

20 10 0 0

20

40

60

80

100

120

Time (months) Fig. 1 e KaplaneMeier estimate of locoregional control by tumour site. The comparison by Log-rank test showed a significant difference in locoregional control for larynx and oropharynx tumours as compared with the other tumour sites (P ! 0.001).

Cells respond to hypoxia by regulating the expression of many genes and the induction of subsequent pathways. By targeting the early steps in the activation of these pathways, more specific and effective therapies can be exploited. One of the main and relatively well-understood hypoxic response pathways involves HIF-1, which has therefore been studied as a tumour-specific target for anticancer treatment [53,59]. Several approaches exist for inhibiting the HIF-1 pathway, aimed at attacking the pathways responsible for

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HIF-1 synthesis, targeting HIF-1 directly, or targeting relevant downstream pathways activated by HIF-1 [12]. The direct inhibition of HIF-1 may occur through modification of the HIF-1a subunit, thereby affecting its activity or stability [12,59]. The expression of HIF-1 can also be regulated independently of hypoxia through pathways frequently altered during carcinogenesis. Recent interest has focused on effectively changing the expression of growth factors, their receptors and pathways that influence the synthesis of HIF-1a protein. Several inhibitors against the epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways and their downstream protein kinases, such as mTOR, are currently under investigation or have already shown therapeutic efficacy [59e61]. Of recent interest, but still unexploited, is a homologous member of the HIF family, HIF-2. Both factors, HIF-1 and HIF-2, are regulated in a similar way, but differ in their transactivation domains, implying that they may regulate distinct target genes [62,63]. Hypoxia response pathways are complicated and offer many options for anticancer treatment. However, whether they have clinical usefulness needs to be confirmed in future research. Although HIF-1 is a good example of a biological response pathway to hypoxia, there are probably numerous unknown molecular responses to the hypoxic environment that act independent of HIF-1. Continued research will undoubtedly unravel this complex network and contribute to novel hypoxia-based treatment modalities.

Patient Selection Treatment strategies have been developed to counteract tumour resistance mechanisms, although usually at the cost of increased short- and long-term side-effects. To maximise the probability that patients benefit from treatments especially designed to modify tumour hypoxia and to provide the best attainable quality of life for individual patients, it is of great importance to develop tools allowing better selection of patients before treatment. It is known from experimental and human studies that there is large heterogeneity in biological characteristics between tumours of the same site and histology, leading to different responses to therapy [6,43]. For validation, the ideal approach would be to incorporate candidate predictive assays in randomised clinical trials. However, it is often difficult to implement these assays on a wide scale in multiple centres with reliable tools to assess tumour oxygenation because these require specific technology and expertise. A randomised trial of continuous hyperfractionated accelerated radiotherapy (CHART) in head and neck squamous cell carcinomas investigated the predictive potential of molecular marker profiles, although they did not measure tumour oxygenation status [64]. They found that three distinct protein expression profiles correlated with different clinical phenotypes, including good locoregional control, poor locoregional control and survival. Later analysis in the CHART trial assessed by Koukourakis et al.

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[63] showed that the expression of endogenous markers of two separate hypoxia response pathways, HIF-2a and carbonic anhydrase IX (CA-IX), was associated with poor locoregional control. Another example is the DAHANCA 5 randomised trial in which a high plasma concentration of osteopontin was found to be associated with poor survival in patients with head and neck cancer [65]. It was concluded that high osteopontin concentrations might predict clinically relevant tumour hypoxia and identify patients who might benefit most from hypoxia-modifying treatments. These studies showed the potential of combining different hypoxia markers in a predictive assay and how different expression patterns of the markers relate to differences in treatment response.

Measurement of Tumour Hypoxia The observations that linked the existence of hypoxia with a more malignant tumour behaviour have elicited numerous studies to establish the prognostic significance of hypoxia for treatment outcome. The ultimate aim of these studies was to provide a good basis for treatment selection dependent on tumour oxygenation. Therefore, the ability to determine the degree and extent of hypoxia in solid tumours is not only important prognostically but also in the selection of patients for hypoxia-modifying treatments. Direct and robust measurements of tumour oxygenation were not possible until the introduction of the polarographic needle electrodes in the 1980s. Use of the needle electrodes clearly showed the clinical relevance of tumour hypoxia. A disadvantage, however, is the restricted use to accessible tumours. Later, alternative methods to assess tumour hypoxia, such as the use of exogenous and endogenous hypoxia markers, were introduced. This enabled the measurement of tumour oxygenation status in potentially all solid tumours and, maybe even more important, the presence of hypoxia could be related to the histological architecture of the tumour. Of great importance is to validate these markers as definitive and useful assays of hypoxia. Clinically very relevant approaches are radiological and nuclear medicine imaging techniques for assessment of the tumour oxygenation status.

Oxygen Electrodes Direct real-time measurement of oxygen tension in tissues can be carried out with polarographic needle electrodes. The fine-needle electrode consumes small amounts of oxygen and is automatically moved through tissues for rapid sampling of oxygen concentrations at multiple points in a tumour [66,67]. Measurements made with the oxygen electrodes provided the first direct evidence for the presence of hypoxia in human cancers [2]. The electrodes have been used to measure pO2 distributions in a large number of experimental and human tumours, showing the heterogeneity of tumour oxygenation status [68e70]. Clinical studies showed that this method could be used to predict tumour response and treatment outcome. These

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reports clearly linked hypoxia measured with fine-needle electrodes to poor patient outcome in squamous cell carcinomas of the head and neck [7], uterine cervix [71] and soft tissue sarcomas [72]. Unexpectedly, one of the latest reports by Nordsmark et al. [73] in human cervix carcinomas, pooling data from three centres, was in disagreement with previous studies. Analysis of the data could not show any prognostic value of tumour hypoxia by polarographic electrode measurements. This may partly be explained by the heterogeneity of the study sample, with relatively small contributions to the study population from each centre and patients treated differently [73]. The oxygen measurements have provided valuable information about the behaviour of human malignancies and for a long time the oxygen electrode technique has been considered the gold standard for assessing tumour oxygenation in the clinic [67,74]. Although a general advantage of the electrode system is the direct and rapid measurement of oxygen tensions, use of the electrodes also has a number of limitations. These include their applicability only to easily accessible tumours and the failure to distinguish necrosis with very low pO2 from severe hypoxia in viable tissue. Furthermore, oxygen electrodes cannot provide information about temporal changes in oxygen tension nor can they give information about patterns of hypoxia that may be important in determining the efficacy of anticancer treatments [67,74,75].

patients with head and neck cancer [82,83] and non-small cell lung cancer [76] showed the potential prognostic value of hypoxia imaging with 18F-MISO for radiotherapy outcome. Important advantages of imaging hypoxia with 18F-MISO PET scanning are that it can be used repeatedly on an almost daily basis due to the short half-life of the tracer and that the complete tumour can be investigated while in situ, instead of single biopsies [81]. A limitation of PET is the low spatial resolution relative to other imaging modalities. DCE MRI is used both experimentally and clinically to monitor the functionality of the tumour vasculature after a contrast agent, gadolinium-DTPA, has been given. Due to the high spatial resolution of MRI heterogeneities in blood flow, the vascular volume and permeability of blood vessels within a tumour can be detected [84]. In a small study of patients with head and neck cancer, this method was used to assess tumour perfusion before and after radiotherapy [85]. Durable local controls were seen mainly in those tumours with a diminished perfusion at the post-radiotherapy assessment. Another study of head and neck carcinomas using the blood oxygen level-dependent effect showed that this MRI technique enables the assessment of improved tumour blood oxygenation by carbogen breathing [86]. These preliminary clinical results indicate that PET and DCE MRI can become important clinical tools for determining vascular function and hypoxia in vivo and for monitoring the effect of therapeutic agents.

Radiological and Nuclear Medicine Imaging Techniques

Exogenous Markers of Tumour Hypoxia

Alternative and non-invasive methods to obtain information about the oxygenation status and vascularisation of human tumours include the use of radiological and nuclear medicine imaging techniques, such as positron emission tomography (PET) using specific tracers and dynamic contrast-enhanced magnetic resonance imaging (DCE MRI). Now, whole-body [18F]-fluorodeoxyglucose (18F-FDG) PET imaging is routinely used for cancer detection, staging and monitoring of response in several tumour types. Although glucose utilisation is indirectly related to the proliferative activity and the oxygenation status of the tumour, 18F-FDG uptake at best correlates weakly with these aspects of tumour biology and more specific radiopharmaceuticals are currently available. A few small clinical studies assessed the value of these tracers and showed several discrepancies indicating that hypoxia and glycolysis do not always correlate [76,77]. The subject of interest during the past few years has been the development of other PET tracers for detecting the proportion of hypoxic cells in vivo. The most widely used is the 2-nitroimidazole 18F-labelled fluoromisonidazole (18F-MISO) [78]. Several animal and human studies evaluated the use of radiolabelled nitroimidazoles for the assessment of the oxygenation status of solid tumours [79e82]. Not only could the presence of hypoxic regions in xenograft tumour lines and human tumours be shown, but also large intra- and inter-tumour variations were observed, even in tumours of the same and different histologies. Recently, studies carried out in

As alternative approaches to polarographic needle electrodes, exogenous hypoxia markers have been developed. These markers are applicable in all solid tumours and provide information about the tumour microenvironment that may be important for treatment outcome. Exogenous hypoxia markers are drugs, chemicals or even bacteria that, after being given to the patient, specifically accumulate or are bioreducible under hypoxic conditions. Clinically relevant markers are the 2-nitroimidazoles pimonidazole and EF5 [87,88]. These agents are injected intravenously before a biopsy is taken or before surgery and have the same mechanism of action. 2-Nitroimidazoles are reductively activated and form protein adducts in mammalian cells at pO2 % 10 mmHg [87,89]. A major advantage of this technique is that dead cells do not generate a signal, as the compounds are only metabolised in viable and metabolically active cells. This could partly explain the lack of correlation between the oxygen electrodes and the exogenous hypoxia markers [88,90]. The electrodes measure oxygen content over a substantially wider sampling range of cells, not distinguishing between viable and dead cells [90]. With immunohistochemical staining techniques it is possible to determine the distribution of hypoxia as indicated by the number of cells positive for pimonidazole or EF5 binding (hypoxic fraction). In a variety of tumours it has allowed the analysis of patterns of hypoxia and the relationships between hypoxia, vasculature, proliferation and other microenvironmental factors [48,91e94]. In

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biopsy material from patients with head and neck squamous cell carcinomas, the vascular and hypoxic parameters were quantified using a computerised image analysis system. Different patterns of hypoxia were described, with pimonidazole staining usually found at a distance from the blood vessels and in perinecrotic areas (Fig. 2) [91]. In the clinical setting, use of the nitroimidazole-binding technique to study the prognostic significance of hypoxia is increasing [6,73,93,95]. A study by Kaanders et al. [6] using biopsy material from patients with advanced head and neck cancer showed worse locoregional control and disease-free survival for patients with high pimonidazole binding as compared with patients with little or no pimonidazole positivity. It was further shown that this association disappeared when patients were treated with ARCON. This strongly suggests that pimonidazole binding indeed reflects hypoxic radiation resistance and may therefore provide a selection tool for hypoxia-modifying treatments on an individual patient basis [6]. However, a recent report could not show the same for patients with cervical carcinomas [73]. This does not mean that pimonidazole can be dismissed, but only encourages further investigations to readdress hypotheses.

Endogenous Markers of Hypoxia Hypoxia-inducible factor-1 The intravenous administration, the inability to investigate archived material and the cost of exogenous hypoxia markers are major limiting factors for their widespread application in the clinic. Attractive alternatives are therefore hypoxia-related genes and proteins as potential endogenous markers. In most cases, transactivation of the genes coding for these proteins is regulated by the transcription factor HIF-1 [96]. HIF-1 is a heterodimer

Fig. 2 e A fluorescent image of a biopsy of larynx carcinoma after injection of the hypoxia marker pimonidazole stained by immunofluorescence for blood vessels (white) and pimonidazole binding (green). Note the hypoxia at some distance from the vessels and necrosis (N) at an even greater distance.

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composed of two subunits, HIF-1a and HIF-1b [96]. Regulation by oxygen is through the HIF-1a subunit, which is rapidly inactivated under normal oxygen conditions by proteasomal degradation [96]. This is achieved via a posttranslational modification of the a subunit that enables the von HippeleLindau protein to bind and target HIF-1a to the proteasome. Under hypoxia, the proteolytic process is suppressed, leading to the up-regulation of HIF-1a, the consecutive activation of HIF-1 and, ultimately, the activation of transcription of many genes [97]. Another homologous member of the same family is HIF-2, which is controlled in a similar way, but regulates different pathways [62,63]. It has been shown that HIF-1, and not HIF-2, regulates glycolysis and the expression of CA-IX [62]. A critical step in the response to hypoxia is induction of the a subunit. It is therefore the primary determinant of HIF activity. Immunohistochemical analysis of human tumour biopsies showed that the overexpression of HIF-1a and HIF-2a is common in solid tumours and their metastases [63,98,99]. The overexpression of both HIF-1a and HIF-2a has been associated with poor locoregional control and overall survival in head and neck carcinomas [63,100], but this could not be shown for carcinomas of the uterine cervix [101]. Furthermore, HIF-1a expression did not correlate with pimonidazole-positive cells or oxygen tensions measured with oxygen electrodes [102,103]. Given this absence of good correlations, it is debatable whether HIF-1a immunohistochemistry is a reliable and useful assay of hypoxia. HIF-2a is still under investigation. Hypoxia-inducible factor-1 target genes In response to low oxygen, HIF-1 is known to transactivate more than 60 genes whose protein products play critical roles in the cellular adaptation to hypoxia [59]. These proteins are involved in many processes, such as angiogenesis, pH regulation, cell proliferation, cell survival and apoptosis, erythropoiesis and energy and glucose metabolism [59]. With the knowledge that many genes are HIF-1 targets, much attention has focussed on their protein products as endogenous markers of tumour hypoxia. These include CA-IX, the glucose transporters 1 and 3 (GLUT-1, GLUT-3) and EPO and its receptor (EPOR). The family of carbonic anhydrases catalyses the reversible hydration of carbon dioxide to carbonic acid, thereby maintaining a stable intracellular pH at the cost of acidification of the extracellular environment [104]. The tumour-associated CA-IX may be particularly important in this function and may play a role in the development of tumours towards a more malignant phenotype [105]. High expression levels of CA-IX have been found in many different cancer cell lines and tumour tissues [105]. In vitro and in vivo studies showed that CA-IX was strongly induced by hypoxia in a broad range of tumours and that expression was mainly found in perinecrotic and poorly perfused tumour areas [6,106e109]. Contradicting observations have been reported on the correlation between tumour hypoxia measured with polarographic needle electrodes and the extent of CA-IX expression [110,111].

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The latest study by Mayer et al. [111] could not find a positive correlation, probably due to other factors that modulate the in vivo CA-IX expression. Considerable colocalisation with pimonidazole staining was observed, although correlations were weak and areas of mismatch were also found (Fig. 3) [6,106]. Several clinical studies in carcinomas of the head and neck, uterine cervix and breast correlated CA-IX expression with (chemo)radiotherapy outcome and found a poor prognosis in patients with high expression levels [109,110,112]. However, also on this subject, results are equivocal and CA-IX alone was not found to be able to predict response to oxygenationmodifying treatment [6]. In the family of glucose transporters, GLUT-1 and GLUT-3 seemed to be the predominant, tumour-expressed members. Increased expression enables a higher cellular uptake of glucose and facilitates anaerobic glycolysis [74]. The relationship with hypoxia has not been investigated, but immunohistochemical staining demonstrated GLUT-1 and GLUT-3 expression at a distance from vessels and adjacent to necrosis [113,114]. In carcinomas of the uterine cervix, GLUT-1 expression has been correlated to oxygen electrode measurements, pimonidazole binding and CA-IX, showing a significant relationship [115,116]. In addition, GLUT-1 and GLUT-3 expression have been associated with disease-free, metastasis-free and overall survival in head and neck carcinomas and carcinomas of the uterine cervix and bladder [114e118]. EPO was long considered to be exclusively produced in the kidney as a specific regulator of erythropoiesis. It became clear that EPO and its receptor (EPOR) are also

expressed in various non-erythroid tissues and in many human tumours [119e122]. It has been suggested that the expression of EPO and its receptor may play a role in the development of hypoxia and therefore a more malignant tumour. The exact relationship has not yet been revealed, but unexpected results from two randomised trials have strengthened these suggestions [123,124]. Both studies reported worse survival and in the head and neck study worse locoregional control in patients receiving rhEPO (epoetin) compared with those on placebo. From these results it was concluded that EPO signalling may have promoted cancer progression and contributed to worse outcome in the experimental arms. In vitro and in vivo studies showed high EPO and EPOR expression under severe hypoxic conditions and in colocalisation with pimonidazolepositive areas [122,125]. However, in contradiction is another study in head and neck cancer biopsies where no colocalisation between EPOR and pimonidazole could be found [126]. The current interest in tumour hypoxia has provoked many studies searching for potential future hypoxia marker candidates. The identification of hypoxia-regulated genes and proteins is not only carried out with immunohistochemistry, but also by DNA, proteomic or tissue array profiling. From previous studies, CA-IX is probably the most promising marker of hypoxia, although its clinical value as a predictive factor has not yet been established. Whether other markers, such as HIF-1, GLUT-1, GLUT-3 and EPO, are useful as markers of hypoxia is still questionable and under investigation. Other hypoxia-related factors that are assumed to be up-regulated under hypoxic stress include osteopontin [127], EGFR [128], involucrin [129] and lactate dehydrogenase-5 [130]. However, these factors are also involved in many other biological processes and they need to be further tested for their specificity as hypoxia markers.

Conclusion

Fig. 3 e A fluorescent image of a biopsy of larynx carcinoma after injection of the hypoxia marker pimonidazole stained by immunofluorescence for blood vessels (white), pimonidazole binding (green) and carbonic anhydrase IX (CA-IX; red). Pimonidazole binding and CA-IX expression are seen at some distance from the vessels with partial colocalisation. CA-IX expression closer to the blood vessels and in pimonidazole-negative areas indicates that CA-IX up-regulation and pimonidazole binding occur at different pO2 levels.

Tumour hypoxia has, for many years, been the subject of investigation, as it plays a critical role in treatment resistance. Successful approaches have been developed to counteract tumour hypoxia, although most of these treatments are accompanied by an increase in side-effects. Thus far, not one treatment targeting tumour hypoxia is widely accepted in clinical practice, but several phase III trials are currently investigating new strategies. For these treatments to be successful it is important to provide a better selection of patients and a better prediction of tumour response by pre-treatment testing of microregional parameters. New methods for qualitative and quantitative assessment of hypoxia demonstrated the prognostic significance and identified candidate markers for future use in predictive assays. Individual markers and also combinations of hypoxia markers have already shown their potential in predicting treatment response. Whether these profiles can be used in the clinic for the customised design of treatment for individual patients remains to be investigated in prospective trials comparing standard treatment against

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experimental treatments targeting the relevant microregional factors. Acknowledgement. This study was supported by grant KUN 2003-2899 of the Dutch Cancer Society. Author for correspondence: I. J. Hoogsteen, Department of Radiation Oncology, 874, Radboud University, Nijmegen Medical Centre, Geert Grooteplein 32, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: [email protected] Received 1 February 2007; accepted 2 March 2007

References 1 Thomlinson RH, Gray LH. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer 1955;9:539e549. 2 Ho ¨ckel M, Schlenger K, Knoop C, Vaupel P. Oxygenation of carcinomas of the uterine cervix: evaluation by computerized O2 tension measurements. Cancer Res 1991;51:6098e6102. 3 Vaupel P, Mayer A, Ho ¨ckel M. Tumor hypoxia and malignant progression. Meth Enzymol 2004;381:335e354. 4 Fyles AW, Milosevic M, Wong R, et al. Oxygenation predicts radiation response and survival in patients with cervix cancer. Radiother Oncol 1998;48:149e156. 5 Knocke TH, Weitmann HD, Feldmann HJ, Selzer E, Po ¨tter R. Intra-tumoral pO2-measurements as predictive assay in the treatment of carcinoma of the uterine cervix. Radiother Oncol 1999;53:99e104. 6 Kaanders JHAM, Wijffels KIEM, Marres HAM, et al. Pimonidazole binding and tumor vascularity predict for treatment outcome in head and neck cancer. Cancer Res 2002;62: 7066e7074. 7 Nordsmark M, Bentzen SM, Rudat V, et al. Prognostic value of tumor oxygenation in 397 head and neck tumors after primary radiation therapy. An international multi-center study. Radiother Oncol 2005;77:18e24. 8 Teicher BA. Hypoxia and drug resistance. Cancer Metastasis Rev 1994;13:139e168. 9 Erler JT, Cawthorne CJ, Williams KJ, et al. Hypoxia-mediated down-regulation of bid and bax in tumors occurs via hypoxiainducible factor 1-dependent and -independent mechanisms and contributes to drug resistance. Mol Cell Biol 2004;24: 2875e2889. 10 Harrison L, Blackwell K. Hypoxia and anemia: factors in decreased sensitivity to radiation therapy and chemotherapy. Oncologist 2004;9(Suppl. 5):31e40. 11 Durand RE. Keynote address: The influence of microenvironmental factors on the activity of radiation and drugs. Int J Radiat Oncol Biol Phys 1991;20:253e258. 12 Wouters BG, Beucken van den T, Magagnin MG, Lambin P, Koumenis C. Targeting hypoxia tolerance in cancer. Drug Resistance Updates 2004;7:25e40. 13 Le QT, Denko NC, Giacca AJ. Hypoxic gene expression and metastasis. Cancer Metastasis Rev 2004;23:293e310. 14 Kaanders JHAM, Pop LAM, Marres HAM, et al. ARCON: experience in 215 patients with advanced head-and-neck cancer. Int J Radiat Oncol Biol Phys 2002;52:769e778. 15 Saunders M, Dische S. Clinical results of hypoxic cell radiosensitisation from hyperbaric oxygen to accelerated radiotherapy, carbogen and nicotinamide. Br J Cancer 1996;27(Suppl.): S271eS278.

393

16 Overgaard J, Hansen HS, Overgaard M, et al. A randomized double-blind phase III study of nimorazole as a hypoxic radiosensitizer of primary radiotherapy in supraglottic larynx and pharynx carcinoma. Results of the Danish Head and Neck Cancer Study (DAHANCA) protocol 5-85. Radiother Oncol 1998; 46:135e146. 17 Ho ¨ckel M, Vaupel P. Tumor hypoxia: definitions and current clinical, biological and molecular aspects. J Natl Cancer Inst 2001;93:266e276. 18 Vaupel P, Thews O, Hoeckel M. Treatment resistance of solid tumors. Role of hypoxia and anemia. Med Oncol 2001;18: 243e259. 19 Harris AL. Hypoxiada key regulatory factor of in tumour growth. Nat Rev Cancer 2002;2:38e47. 20 Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res 1989;49:6449e6465. 21 Dewhirst MW, Ong ET, Braun RD, et al. Quantification of longitudinal tissue pO2 gradients in window chamber tumours: impact on tumour hypoxia. Br J Cancer 1999;79:1717e1722. 22 Wouters BG, Brown JM. Cells at intermediate oxygen levels can be more important than the ‘‘hypoxic fraction’’ in determining tumor response to fractionated radiotherapy. Radiat Res 1997; 147:541e550. 23 Gray LH, Conger AD, Ebert M, Hornsey S, Scott OC. The concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Br J Radiol 1953;26: 638e648. 24 Horsman MR, Overgaard J. The oxygen effect and tumour microenvironment. In: Steel GG, editor. Basic clinical radiobiology. London: Arnold; 2002. p. 158e168. 25 Isa AY, Ward TH, West CML, Slevin NJ, Homer JJ. Hypoxia in head and neck cancer. Br J Radiol 2006;79:791e798. 26 Ho ¨ckel M, Schlenger K, Aral B, Mitze M, Scha ¨ffer U, Vaupel P. Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res 1996;56: 4509e4515. 27 Bennewith KL, Durand RE. Quantifying transient hypoxia in human tumor xenografts by flow cytometry. Cancer Res 2004; 64:6183e6189. 28 Kirkpatrick JP, Cardenas-Navia LI, Dewhirst MW. Predicting the effect of temporal variations in pO2 on tumor radiosensitivity. Int J Radiat Oncol Biol Phys 2004;59:822e833. 29 Belle van SJP, Cocquyt V. Impact of haemoglobin levels on the outcome of cancers treated with chemotherapy. Crit Rev Oncol/Hematol 2003;47:1e11. 30 Knight K, Wade S, Balducci L. Prevalence and outcome of anemia in cancer: a systematic review of the literature. Am J Med 2004;116:11Se26S. 31 Becker A, Stadler P, Lavey RS, et al. Severe anemia is associated with poor tumor oxygenation in head and neck squamous cell carcinomas. Int J Radiat Oncol Biol Phys 2000;46:459e466. 32 Hirst DG. What is the importance of anaemia in radiotherapy? The value of animal studies. Radiother Oncol 1991;(Suppl. 20):29e33. 33 Kelleher DK, Matthiensen U, Thews O, Vaupel P. Blood flow, oxygenation, and bioenergetic status of tumors after erythropoietin treatment in normal and anemic rats. Cancer Res 1996; 56:4728e4734. 34 Dunst J, Kuhnt T, Strauss HG, et al. Anemia in cervical cancers: impact on survival, patterns of relapse, and association with hypoxia and angiogenesis. Int J Radiat Oncol Biol Phys 2003; 56:778e787. 35 Vaupel P, Mayer A, Briest S, Hoeckel M. Oxygenation gain factor: a novel parameter characterizing the association

394

36

37

38

39

40

41

42

43

44

45

46

47 48

49

50

51

52

CLINICAL ONCOLOGY between hemoglobin level and the oxygenation status of breast cancers. Cancer Res 2003;63:7634e7637. Nordsmark M, Overgaard J. Tumor hypoxia is independent of hemoglobin and prognostic for loco-regional tumor control after primary radiotherapy in advanced head and neck cancer. Acta Oncol 2004;43:396e403. Koukourakis MI, Giatromanolaki A, Sivridis E, et al. Hypoxiamediated tumor pathways of angiogenesis and pH regulation independent of anemia in head-and-neck cancer. Int J Radiat Oncol Biol Phys 2004;59:67e71. Winter SC, Shah KA, Han C, et al. The relation between hypoxia-inducible factor (HIF)-1a and HIF-2a expression with anemia and outcome in surgically treated head and neck cancer. Cancer 2006;107:757e766. Overgaard J, Horsman MR. Modification of hypoxia-induced radioresistance in tumors by the use of oxygen and sensitizers. Semin Radiat Oncol 1996;6:10e21. Kaanders JHAM, Bussink J, van der Kogel AJ. Clinical studies of hypoxia modification in radiotherapy. Semin Radiat Oncol 2004;14:233e240. Kaanders JHAM, Bussink J, Kogel van der AJARCON. a novel biology-based approach in radiotherapy. Lancet Oncol 2002;3: 728e737. Rojas A, Hirst VK, Calvert AS, Johns H. Carbogen and nicotinamide as radiosensitizers in a murine mammary carcinoma using conventional and accelerated radiotherapy. Int J Radiat Oncol Biol Phys 1996;34:357e365. Bussink J, Kaanders JHAM, Rijken PFJW, et al. Vascular architecture and microenvironmental parameters in human squamous cell carcinoma xenografts: effects of carbogen and nicotinamide. Radiother Oncol 1999;50:173e184. Bernier J, Denekamp J, Rojas A, et al. ARCON: accelerated radiotherapy with carbogen and nicotinamide in non small cell lung cancer: a phase I/II study by the EORTC. Radiother Oncol 1999;52:149e156. Hoskin PJ, Saunders MI, Dische S. Hypoxic radiosensitizers in radical radiotherapy for patients with bladder carcinoma. Cancer 1999;86:1322e1328. Miralbell R, Mornex F, Greiner R, et al. Accelerated radiotherapy, carbogen, and nicotinamide in glioblastoma multiforme: a report of European Organization for Research and Treatment of Cancer trial 22933. J Clin Oncol 1999;17:3143e3149. Bernsen HJ, Rijken PF, Peters H, et al. Hypoxia in a human intracerebral glioma model. J Neurosurg 2000;93:449e454. Hoskin PJ, Sibtain A, Daley FM, Saunders MI, Wilson GD. The immunohistochemical assessment of hypoxia, vascularity and proliferation in bladder carcinoma. Radiother Oncol 2004;72: 159e168. Hoogsteen IJ, Pop LAM, Marres HAM, et al. Oxygen-modifying treatment with ARCON reduces the prognostic significance of hemoglobin in squamous cell carcinoma of the head and neck. Int J Radiat Oncol Biol Phys 2006;64:83e89. Mendenhall WM, Morris CG, Andur RJ, Mendenhall NP, Siemann DW. Radiotherapy alone or combined with carbogen breathing for squamous cell carcinoma of the head and neck. A prospective, randomized trial. Cancer 2005;104:323e337. Hoskin PJ, Rojas AM, Phillips H, Saunders MI. Acute and late morbidity in the treatment of advanced bladder carcinoma with accelerated radiotherapy, carbogen, and nicotinamide. Cancer 2005;103:2287e2297. Overgaard J, Hansen HS, Andersen AP, et al. Misonidazole combined with split-course radiotherapy in the treatment of invasive carcinoma of larynx and pharynx: report from the DAHANCA 2 study. Int J Radiat Oncol Biol Phys 1989;16:1065e1068.

53 Wouters BG, Weppler SA, Koritzinsky M, et al. Hypoxia as a target for combined modality treatments. Eur J Cancer 2002; 38:240e257. 54 Janssen HL, Haustermans KM, Balm AJ, Begg AC. Hypoxia in head and neck cancer: how much, how important? Head and Neck 2005;27:622e638. 55 Brown JM, Lemmon MJ. Potentiation by the hypoxic cytotoxin SR 4233 of cell killing produced by fractionated irradiation of mouse tumors. Cancer Res 1990;50:7745e7749. 56 Dorie MJ, Brown JM. Tumor-specific, schedule-dependent interaction between tirapazamine (SR 4233) and cisplatin. Cancer Res 1993;53:4633e4636. 57 von Pawel J, von Roemeling R, Gatzemeier U, et al. Tirapazamine plus cisplatin versus cisplatin in advanced non-smallcell lung cancer: a report of the international CATAPULT I study group. J Clin Oncol 2000;18:1351e1359. 58 Rischin D, Peters L, Fisher R, et al. Tirapazamine, cisplatin, and radiation versus fluorouracil, cisplatin, and radiation in patients with locally advanced head and neck cancer: a randomized phase II trial of the Trans-Tasman Radiation Oncology Group (TROG 98.02). J Clin Oncol 2005;23:79e87. 59 Semenza GL. Targeting HIF-1 for cancer therapy. Nature Rev Cancer 2003;3:721e732. 60 Hynes NE, Lane HA. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 2005;5:341e354. 61 Shaw RJ, CantleyRas LC. PI(3)K and mTOR signalling controls tumour cell growth. Nature 2006;44:424e430. 62 Hu CJ, Wang LY, Chodosh LA, Keith B, Simon MC. Differential roles of hypoxia-inducible factor 1a (HIF-1a) and HIF-2a in hypoxic gene regulation. Mol Cell Biol 2003;23:9361e9374. 63 Koukourakis MI, Bentzen SM, Giatromanolaki A, et al. Endogenous markers of two separate hypoxia response pathways (hypoxia inducible factor 2 alpha and carbonic anhydrase 9) are associated with radiotherapy failure in head and neck cancer patients recruited in the CHART randomized trial. J Clin Oncol 2006;24:727e735. 64 Buffa F, Bentzen SM, Daley FM, et al. Molecular marker profiles predict locoregional control of head and neck squamous cell carcinoma in a randomized trial of continuous hyperfractionated accelerated radiotherapy. Clin Cancer Res 2004;10:3745e3754. 65 Overgaard J, Eriksen JG, Nordsmark M, Alsner J, Horsman MR. Plasma osteopontin, hypoxia, and response to the hypoxia sensitiser nimorazole in radiotherapy of head and neck: results from the DAHANCA 5 randomised double-blind placebocontrolled trial. Lancet Oncol 2005;6:757e764. 66 Seddon BM, Honess DJ, Vojnovic B, Tozer GM, Workman P. Measurement of tumor oxygenation: in vivo comparison of a luminescence fiber-optic sensor and a polarographic electrode in the p22 tumor. Radiat Res 2001;155:837e846. 67 Milosevic M, Fyles A, Hedley D, Hill R. The human tumor microenvironment: invasive (needle) measurement of oxygen and interstitial fluid pressure. Semin Radiat Oncol 2004;14: 249e258. 68 Lyng H, Sundfør K, Rofstad EK. Oxygen tension in human tumours measured with polarographic needle electrodes and its relationship to vascular density, necrosis and hypoxia. Radiother Oncol 1997;44:163e169. 69 Adam MF, Gabalski EC, Bloch DA, et al. Tissue oxygen distribution in head and neck cancer patients. Head Neck 1999;21:146e153. 70 Le QT, Kovacs MS, Dorie MJ, et al. Comparison of the comet assay and the oxygen microelectrode for measuring tumor oxygenation in head-and-neck cancer patients. Int J Radiat Oncol Biol Phys 2003;56:375e383.

TUMOUR HYPOXIA AND HYPOXIA MODIFICATION 71 Fyles AW, Milosevic M, Hedley D, et al. Tumor hypoxia has independent predictor impact only in patients with nodenegative cervix cancer. J Clin Oncol 2002;20:680e687. 72 Brizel DM, Scully SP, Harrelson JM, et al. Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res 1996;56:941e943. 73 Nordsmark M, Loncaster J, Aquino-Parsons C, et al. The prognostic value of pimonidazole and tumour pO2 in human cervix carcinomas after radiation therapy: a prospective international multi-center study. Radiother Oncol 2006;80:123e131. 74 Williams KJ, Parker CA, Stratford IJ. Exogenous and endogenous markers of tumour oxygenation status. Definitive markers of tumour hypoxia? Adv Exp Med Biol 2005;566:285e294. 75 Evans SM, Koch CJ. Prognostic significance of tumor oxygenation in humans. Cancer Lett 2003;195:1e16. 76 Eschmann SM, Paulsen F, Reimold M, et al. Prognostic impact of hypoxia imaging with 18F-misonidazole PET in non-small cell lung cancer and head and neck cancer before radiotherapy. J Nucl Med 2005;46:253e260. 77 Thorwarth D, Eschmann SM, Holzner F, Paulsen F, Alber M. Combined uptake of [18F]FDG and [18F]FMISO correlates with radiation therapy outcome in head-and-neck cancer patients. Radiother Oncol 2006;80:151e156. 78 Chapman JD, Engelhardt EL, Stobbe CC, Schneider RF, Hanks GE. Measuring hypoxia and predicting tumor radioresistance with nuclear medicine assays. Radiother Oncol 1998; 46:229e237. 79 Dubois L, Landuyt W, Haustermans K, et al. Evaluation of hypoxia in an experimental rat tumour model by [18F] fluoromisonidazole PET and immunohistochemistry. Br J Cancer 2004;91:1947e1954. 80 Lethio ¨ K, Eskola O, Viljanen T, et al. Imaging perfusion and hypoxia with PET to predict radiotherapy response in headand-neck cancer. Int J Radiat Oncol Biol Phys 2004;59: 971e982. 81 Troost EGC, Laverman P, Kaanders JHAM, et al. Imaging hypoxia after oxygenation-modification: comparing [18F]FMISO autoradiography with pimonidazole immunohistochemistry in human xenograft tumors. Radiother Oncol 2006;80:157e164. 82 Rajendran JG, Schwartz DL, O’Sullivan J, et al. Tumor hypoxia imaging with [F-18] fluoromisonidazole positron emission tomography in head and neck cancer. Clin Cancer Res 2006; 12:5435e5441. 83 Hicks RJ, Rischin D, Fisher R, Binns D, Scott AM, Peters LJ. Utility of FMISO PET in advanced head and neck cancer treated with chemoradiation incorporating a hypoxia-targeting chemotherapy agent. Eur J Nucl Med Mol Imaging 2005;32: 1384e1391. 84 Delorme S, Knopp MV. Non-invasive vascular imaging: assessing tumour vascularity. Eur Radiol 1998;8:517e527. 85 Hoskin PJ, Saunders MI, Goodchild K, Powell MEB, Taylor NJ, Baddeley H. Dynamic contrast enhanced magnetic resonance scanning as a predictor of response to accelerated radiotherapy for advanced head and neck cancer. Br J Radiol 1999;72: 1093e1098. 86 Rijpkema M, Kaanders JHAM, Joosten FBM, van der Kogel AJ, Heerschap A. Effects of breathing a hyperoxic hypercapnic gas mixture on blood oxygenation and vascularity of head-andneck tumors as measured by magnetic resonance imaging. Int J Radiat Oncol Biol Phys 2002;53:1185e1191. 87 Raleigh JA, Calkins-Adams DP, Rinker LH, et al. Hypoxia and vascular endothelial growth factor expression in human squamous cell carcinomas using pimonidazole as a hypoxia marker. Cancer Res 1998;58:3765e3768.

395

88 Evans SM, Hahn S, Pook DR, et al. Detection of hypoxia in human squamous cell carcinoma by EF5 binding. Cancer Res 2000;60:2018e2024. 89 Koch CJ, Evans SM, Lord EM. Oxygen dependence of cellular uptake of EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide]: analysis of drug adducts by fluorescent antibodies vs bound radioactivity. Br J Cancer 1995;72: 869e874. 90 Nordsmark M, Loncaster J, Aquino-Parsons C, et al. Measurements of hypoxia using pimonidazole and polarographic oxygen-sensitive electrodes in human cervix carcinomas. Radiother Oncol 2003;67:35e44. 91 Wijffels KIEM, Kaanders JHAM, Rijken PFJW, et al. Vascular architecture and hypoxic profiles in human head and neck squamous cell carcinomas. Br J Cancer 2000;83:674e683. 92 Evans SM, Hahn SM, Magarelli DP, Koch CJ. Hypoxic heterogeneity in human tumors. EF5 binding, vasculature, necrosis and proliferation. Am J Clin Oncol 2001;24:467e472. 93 Carnell DM, Smith RE, Path MRC, et al. An immunohistochemical assessment of hypoxia in prostate carcinoma using pimonidazole: implications for radioresistance. Int J Radiat Oncol Biol Phys 2006;65:91e99. 94 van Laarhoven HWM, Kaanders JHAM, Lok J, et al. Hypoxia in relation to vasculature and proliferation in liver metastases in patients with colorectal cancer. Int J Radiat Oncol Biol Phys 2006;64:473e482. 95 Evans SM, Fraker D, Hanhn SM, et al. EF5 binding and clinical outcome in human soft tissue sarcomas. Int J Radiat Oncol Biol Phys 2006;64:922e927. 96 Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci 1995;92: 5510e5514. 97 Pugh CW, Ratcliffe PJ. The von HippeleLindau tumor suppressor, hypoxia-inducible factor-1 (HIF-1) degradation, and cancer pathogenesis. Semin Cancer Biol 2003;13:83e89. 98 Zhong H, De Marzo AM, Laughner E, et al. Overexpression of hypoxia-inducible factor 1a in common human cancers and their metastases. Cancer Res 1999;59:5830e5835. 99 Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1a and HIF-2a in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol 2000;157:411e421. 100 Aebersold DM, Burri P, Beer KT, et al. Expression of hypoxiainducible factor-1a: a novel predictive and prognostic parameter in the radiotherapy of oropharyngeal cancer. Cancer Res 2001;61:2911e2916. 101 Hutchison GJ, Valentine HR, Loncaster JA, et al. Hypoxiainducible factor 1a expression as an intrinsic marker of hypoxia: correlation with tumor oxygen, pimonidazole measurements, and outcome in locally advanced carcinoma of the cervix. Clin Cancer Res 2004;10:8405e8412. 102 Mayer A, Wree A, Hockel M, et al. Lack of correlation between expression of HIF-1a protein and oxygenation status in identical tissue areas of squamous cell carcinomas of the uterine cervix. Cancer Res 2004;64:5876e5881. 103 Vordermark D, Kraft P, Katzer A, et al. Glucose requirement for hypoxic accumulation of hypoxia-inducible factor-1a (HIF-1a). Cancer Lett 2005;230:122e133. 104 Sly WS, Hu PY. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem 1995;64:375e401. 105 Ivanov S, Liao SY, Ivanova A, et al. Expression of hypoxiainducible cell-surface transmembrane carbonic anhydrases in human cancer. Am J Pathol 2001;158:905e919.

396

CLINICAL ONCOLOGY

106 Wykoff CC, Beasley N, Watson PH, et al. Hypoxia-inducible expression of tumor associated carbonic anhydrases. Cancer Res 2000;60:7075e7083. 107 Lal A, Peters H, St Croix B, et al. Transcriptional response to hypoxia in human tumors. J Natl Cancer Inst 2001;93: 1337e1343. 108 Olive PL, Aquino-Parsons C, MacPhail SH, et al. Carbonic anhydrase 9 as an endogenous marker for hypoxic cells in cervical cancer. Cancer Res 2001;61:8924e8929. 109 Koukourakis MI, Giatromanolaki A, Sivridis E, et al. Hypoxiaregulated carbonic anhydrase-9 (CA9) relates to poor vascularization and resistance of squamous cell head and neck cancer to chemoradiotherapy. Clin Cancer Res 2001;7: 3399e3403. 110 Loncaster JA, Harris AL, Davidson SE, et al. Carbonic anhydrase (CA IX) expression, a potential new intrinsic marker of hypoxia: correlations with tumor oxygen measurements and prognosis in locally advanced carcinoma of the cervix. Cancer Res 2001;61:6394e6399. 111 Mayer A, Ho ¨ckel M, Vaupel P. Carbonic anhydrase IX expression and tumor oxygenation status do not correlate at the microregional level in locally advanced cancers of the uterine cervix. Clin Cancer Res 2005;11:7220e7225. 112 Brennan DJ, Jirstom K, Kronblad A, et al. CA IX is an independent prognostic marker in premenopausal breast cancer patients with one to three positive lymph nodes and a putative marker of radiation resistance. Clin Cancer Res 2006;12:6421e6431. 113 Oliver RJ, Woodwards RTM, Sloan P, Thakker NS, Stratford IJ, Airley RE. Prognostic value of facilitative glucose transporter Glut-1 in oral squamous cell carcinomas treated by surgical resection: results of EORTC translational research fund studies. Eur J Cancer 2004;40:503e507. 114 Jonathan RA, Wijffels KIEM, Peeters W, et al. The prognostic value of endogenous hypoxia-related markers for head and neck squamous cell carcinomas treated with ARCON. Radiother Oncol 2006;79:288e297. 115 Airley R, Loncaster J, Davidson S, et al. Glucose transporter GLUT-1 expression correlates with tumor hypoxia and predicts metastasis-free survival in advanced carcinoma of the cervix. Clin Cancer Res 2001;7:928e934. 116 Airley RE, Loncaster J, Raleigh JA, et al. GLUT-1 and CAIX as intrinsic markers of hypoxia in carcinoma of the cervix: relationship to pimonidazole binding. Int J Cancer 2003;104: 85e91. 117 Baer S, Casaubon L, Schwartz MR, Marcogliese A, Younes M. Glut3 expression in biopsy specimens of laryngeal carcinoma is associated with poor survival. Laryngoscope 2002;112:393e396.

118 Palit V, Philips RM, Puri R, Shah T, Bibby MC. Expression of HIF1alpha and GLUT-1 in human bladder cancer. Oncol Rep 2005; 14:909e913. 119 Yasuda Y, Masuda S, Chikuma M, Inoue K, Nagao M, Sasaki R. Estrogen-dependent production of erythropoietin in uterus and its implication in uterine angiogenesis. J Biol Chem 1998; 273:25381e25387. 120 Acs G, Zhang PJ, Rebbeck TR, Acs P, Verma A. Immunohistochemical expression of erythropoietin and erythropoietin receptor in breast carcinoma. Cancer 2002;95:969e981. 121 Genc S, Koroglu TF, Genc K. Erythropoietin and the nervous system. Brain Res 2004;1000:19e31. 122 Arcasoy MO, Amin K, Chou SC, Haroon ZA, Varia M, Raleigh JA. Erythropoietin and erythropoietin receptor expression in head and neck cancer: relationship to tumor hypoxia. Clin Cancer Res 2005;11:20e27. 123 Henke M, Laszig R, Ru ¨be C, et al. Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomized, double-blind, placebo-controlled trial. Lancet 2003;362:1255e1260. 124 Leyland-Jones B, Semiglazov V, Pawlicki M, et al. Maintaining normal hemoglobin levels with epoetin alfa in mainly nonanemic patients with metastatic breast cancer receiving firstline chemotherapy: a survival study. J Clin Oncol 2005;23: 5960e5972. 125 Acs G, Zhang PJ, McGrath CM, et al. Hypoxia-inducible erythropoietin signaling in squamous dysplasia and squamous cell carcinoma of the uterine cervix and its potential role in cervical carcinogenesis and tumor progression. Am J Pathol 2003;162:1789e1806. 126 Hoogsteen IJ, Peeters WJM, Marres HAM, et al. Erythropoietin receptor is not a surrogate marker for tumor hypoxia and does not correlate with survival in head and neck squamous cell carcinomas. Radiother Oncol 2005;76:194e199. 127 Le QT, Sutphin PD, Raychaudhuri S, et al. Identification of osteopontin as a prognostic plasma marker for head and neck squamous cell carcinomas. Clin Cancer Res 2003;9:59e67. 128 Nishi H, Nishi KH, Johnson AC. Early growth response-1 gene mediates up-regulation of epidermal growth factor receptor expression during hypoxia. Cancer Res 2002;62:827e834. 129 Chou S-C, Azuma Y, Varia MA, Raleigh JA. Evidence that involucrin, a marker for differentiation, is oxygen regulated in human squamous cell carcinoma. Br J Cancer 2004;90: 728e735. 130 Koukourakis MI, Giatromanolaki A, Sivridis E, et al. Lactate dehydrogenase-5 (LDH-5) overexpression in non-small-cell lung cancer is linked to tumour hypoxia, angiogenic factor production and poor prognosis. Br J Cancer 2003;89:877e885.